Construction And Evaluation Of Tumor Microenvironment Immune Regulated Nano-micelles For Anti-breast Cancer

Cancer immunotherapy is a revolutionary treatment for cancer therapy with the in-depth study of tumor immunology.The purpose of immunotherapy is to reactivate the immune system,in which the immune response mediated by cytotoxic CD8~+T cells plays a major role anti-tumor immunity.However,T cells and tumor cells express many immune checkpoint molecules,such as programmed death receptor 1(programmed cell death protein,PD-1),cytotoxic T lymphocyte associated antigen 4(cytotoxic T lymphocyte-associatedantigen-4,CTLA-4),which mediate immunosuppression and immune escape.There are still many limitations in clinical application of immune checkpoint blocked which has been proved to have great therapeutic potential.The low response rate of T cells and the complex regulatory network among a variety of immunosuppressive cells make the effect of single target therapy unsatisfactory.Nano delivery system can coordinate drug co-delivery to achieve multi-target regulation.In this study,we reported a poly-peptide-based micellar system that encapsulates aryl hydrocarbon receptor(AhR)inhibitor CH223191 and connected with anti-CD28 by matrix metalloproteinase-2(MMP-2)sensitive peptides,which could be released in tumor microenvironment(TME)to provide costimulatory signals.AhR blocking could down-regulate the PD-1 expression of CD8~+T cells,relieve immunosuppression,as well as reduce the proliferative activity and metastasis of breast cancer cells.Anti-CD28,which could provide the costimulatory signal and effectively improve the efficiency of T cell activation,cooperating with the immunomodulatory effect of AhR,to restore anti-tumor immunity and inhibit tumor growth by multi-pathway synergistic regulation.We systematically evaluated the preparation,immunomodulatory and anti-breast cancer effects of nano-immunoregulatory micelles in vivo and in vitro.The project is mainly divided into three chapters.In first chapter,we constructed and characterized co-loaded immune micelles.Firstly,the polypeptide monomer with sequence stearyl-HHHRRRRRPLGLAGK-(Mal)(SHRP)was synthesized by solid phase synthesis,and its purity and molecular weight were detected and identified.The results showed that the peptide monomer was synthesized successfully and the purity was high enough for subsequent research.Then we prepared the CH223191-encapsulated micellar system SHRP-CH by emulsion solvent evaporation method.In the next step,the anti-CD28 was conjugated to the micelle to form co-loaded immune micelle Dual-SHRP through the reaction between the-Mal of the peptide and the anti-CD28 after thiolation.The critical micelle concentration(CMC)of SHRP is about 0.1μg/m L,which can effectively avoid drug leakage caused by dilution of body fluids.Then we characterized the synthesized micelles.The particle size of the prepared SHRP was about 124nm,which increased to 162nm after drug loading and antibody conjunction.When treated with MMP-2,the enzyme-sensitive peptide fragment was broken and the particle size of the micelle(MMP-Dual)reduced to 88nm.When observed by transmission electron microscope(TEM),SHRP and Dual-SHRP both showed spherical structure observed,while MMP-Dual still maintained the spherical-likely morphology.The drug loading of CH223191 and the antibody conjunction amount of anti-CD28 in Dual-SHRP were(7.18±1.2)%and(5.3±0.37)%,respectively.Finally,the in vitro release of the CH223191 and anti-CD28 were investigated.In second chapter,we had a preliminary study on the biological activity,immunomodulatory effect and anti-breast cancer activity of Dual-SHRP in vitro.First,the immunogenicity of SHRP was investigated,and the results showed that the absence of immunogenicity of SHRP would not cause immune response.The biological function of anti-CD28 was verified by flow cytometry.The results of flow cytometry confirmed that the ability of anti-CD28 binding to CD28 molecule and transmitting costimulatory signals was not affected after conjunction to micelles.The results of cell uptake assay showed that the micelles were more easily internalized by tumor cells and CD8~+T cells.The cellular uptake efficiency of MMP-Dual was higher,which was consistent with the results of anti-migration of 4T1 cells in vitro and PD-1 expression of CD8~+T cells.so that the efficacy of CH223191was improved.Finally,in order to mimic the exhausted state of TME,the CD8~+T cells was first induced into exhaustion in vitro and the co-culture system of exhausted CD8~+T and 4T1cells was constructed.The apoptosis rates of tumor cells were detected after different treatment.The results showed that Dual-SHRP could effectively relieve immunosuppression and restore the tumor killing effect of CD8~+T cells.For Chapter 3,we investigated the anti-tumor effect and immunomodulatory mechanism of Dual-SHRP in vivo.Firstly,the orthotopic transplantation tumor model of breast cancer in mice loaded with 4T1 cells was established.In vivo living imaging results show that Dual-SHRP can accumulate more in the tumor site than free drugs.The tumor volume growth curve and survival test confirmed that Dual-SHRP could effectively inhibit tumor growth in mice.The results of HE staining and blood biochemical indexes confirmed the biosafety of Dual-SHRP.Finally,the immunomodulatory mechanism of Dual-SHRP was investigated,and it was found that Dual-SHRP could effectively increase tumor lymphatic infiltration,increase the number of cytotoxic CD8~+T cells,reduce the proportion of inhibitory immune cell subsets and increase the secretion of anti-tumor cytokines.In conclusion,we constructed a polypeptide-based MMP-2 enzyme-sensitive micelles system Dual-SHRP,which could effectively regulate the suppressive tumor immune microenvironment(tumor immune microenvironment,TIME)and inhibit breast cancer growth.Micelles were accumulated at the tumor site by passive targeting.The in vivo and in vitro results indicated that Dual-SHRP could effectively inhibit the growth of breast cancer in mice,increase the percentage of CD8~+T cells in the tumor,and reduce the proportion of immunosuppressive lymphocytes.This project explored the potential of AhR regulation combined with increasing costimulatory signals in regulating tumor microenvironment immunity.The developed co-loaded micellar system is a new and effective tumor immunotherapy strategy,and also provides a new nano-platform for the delivery of immunomodulators.